Project Summary/Abstract Because of their favorable characteristics, ?-lactams make up approximately 60% of antibiotic usage worldwide. Bacterial resistance to ?-lactam drugs, however, has been steadily increasing, posing a significant threat to antibiotic therapy. The most common mechanism of resistance is ?-lactamase-catalyzed hydrolysis, which renders the antibiotics ineffective. An area of particular concern is multi-drug resistant infections caused by Gram-negative rods; leaving few options for treatment. Carbapenems are a class of ?-lactam antibiotics that historically have been less susceptible to the action of ?-lactamases and have seen increased usage. A rising number of bacterial infections acquired in hospital settings, however, are caused by carbapenem resistant Enterobactericiae (CRE) pathogens. Enterobacteriaceae frequently become resistant to carbapenem antibiotics by acquiring plasmid-encoded ?-lactamases named carbapenemases. The three most frequently encountered carbapenemases in clinics worldwide are KPC-2, NDM-1, and OXA-48. The objective of this application is to utilize DNA-encoded chemical library technology to discover small molecule inhibitors of these enzymes. This approach involves the creation of libraries of drug-like molecules covalently attached to a unique DNA barcode that enables identification of binders for a target in a large pool of compounds. DNA-encoded chemical libraries have been constructed at the Center for Drug Discovery at Baylor College of Medicine that encode over 2 billion compounds, allowing a screen of wide chemical space for novel, non-?-lactam inhibitors of these important drug-resistance enzymes. In preliminary studies, we demonstrated the feasibility of the approach by using a DNA-encoded chemical library to identify CDD-97, which inhibits OXA-48 with a Ki of 600 nM. The X-ray structure of OXA-48 in complex with CDD-97 revealed it makes key interactions with active site residues and medicinal chemistry studies have defined structure-activity relationships for the molecule. DNA-encoded chemical library screens will be extended for the OXA-48, NDM-1 and KPC-2 ?-lactamases and identified inhibitors will be characterized with respect to potency of inhibition, X-ray structure, spectrum of inhibition, and bioactivity versus bacteria. In addition, medicinal chemistry methods will be used to optimize potency and accumulation of inhibitors in bacteria. The proposed experiments have the potential to yield new, non-?-lactam, inhibitors and provide insights into chemical scaffolds that are favorable for interaction with ?-lactamases.